title: Bacterial expression and spectroscopic characterization of soybean leghaemoglobin a.
abstract: A gene encoding leghaemoglobin a from soybean has been constructed and the soluble recombinant protein expressed in E. coli. The integrity of the recombinant protein has been assessed by a range of spectroscopic techniques. Electrospray mass spectrometry of the protein indicates that the molecular mass of the protein corresponds to the predicted amino acid sequence. Circular dichroism spectra of the ferric derivative and UV-visible spectra of various ferric and ferrous derivatives (pH 6.99, mu = 0.10 M, 25.0 degrees C) are consistent with published data for the wild-type protein. For the ferric derivative, UV-visible (298 and 77 K) and EPR (10 K) spectra indicate the existence of a thermal equilibrium between high- and low-spin forms. Titration of the protein (0.10 M NaCl, mu = 0.10 M, 25.0 degrees C) between pHs 6.68 and 10.35 indicate formation (pKa = 8.3+/-0.03) of a 6-coordinate, hydroxide-bound form of the protein at high pH. All of the above data are consistent with the behaviour of the wild-type protein.
authors.affiliation: Department of Chemistry, University of Leicester, University Road, Leicester, LE1 7RH, England, U.K.
authors.forename: D K
authors.initials: DK
authors.lastname: Jones
authors.forename: R
authors.initials: R
authors.lastname: Badii
authors.forename: F I
authors.initials: FI
authors.lastname: Rosell
authors.forename: E
authors.initials: E
authors.lastname: Lloyd
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D005813:Genes, Synthetic; D006863:Hydrogen-Ion Concentration; D007874:Leghemoglobin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis; D013025:Glycine max; D013054:Spectrophotometry, Atomic
chemical_list: D007874:Leghemoglobin; D011994:Recombinant Proteins